Biology Reference
In-Depth Information
15. The sediment of cell debris, nuclei and chloroplasts is tight,
allowing simple decanting of the supernatant. Overlaying the
CE supernatant on top of the Percoll gradients shall be done as
quickly as possible but also carefully. A second pair of helping
hands is advantageous. The CE supernatant fraction shall be
quantitatively loaded onto the Percoll gradients to maximize
yield.
16. If the leaf peroxisomes are very labile, for instance if deriving
from stressed plants or senescent leaves, they tend to lyse dur-
ing ultracentrifugation and fl oat from their typical density of
ca. 1.23 g/mL to the top of the sucrose density gradient.
Hence, shorter centrifugation times (e.g., 40 min) are recom-
mended for labile peroxisomes to obtain maximum yield.
17. Collect the peroxisome band in no more than 1.2 mL because
the viscosity of the sucrose solutions needs to be reduced to
guarantee accurate pipetting and freezing.
18. Protein determination has been carried out according to
Lowry [
19
].
Acknowledgment
Dr. R. Singhal was supported by an YGGDRASIL IS-MOBIL
fellowship from the Norwegian Research Council.
References
1. Yu C, Huang AH (1986) Conversion of serine
to glycerate in intact spinach leaf peroxisomes:
role of malate dehydrogenase. Arch Biochem
Biophys 245:125-133
2. Lopez-Huertas E, Sandalio LM, Del Rio LA
(1995) Integral membrane polypeptides of pea
leaf peroxisomes: characterization and response
to plant stress. Plant Physiol Biochem
33:295-302
3. Cooper TG, Beevers H (1969) Beta oxidation
in glyoxysomes from castor bean endosperm.
J Biol Chem 244:3514-3520
4. Harrison-Lowe N, Olsen LJ (2006) Isolation
of glyoxysomes from pumpkin cotyledons.
Curr Protoc Cell Biol 3(19):1-8
5. The Arabidopsis Genome Initiative (2000)
Analysis of the genome sequence of the fl owering
plant Arabidopsis thaliana. Nature 408:796-815
6. Fukao Y, Hayashi M, Nishimura M (2002)
Proteomic analysis of leaf peroxisomal proteins
in greening cotyledons of Arabidopsis thaliana.
Plant Cell Physiol 43:689-696
7. Reumann S, Babujee L, Ma C et al (2007)
Proteome analysis of Arabidopsis leaf peroxi-
somes reveals novel targeting peptides, meta-
bolic pathways, and defense mechanisms. Plant
Cell 19:3170-3193
8. Reumann S, Quan S, Aung K et al (2009)
In-depth proteome analysis of Arabidopsis leaf
peroxisomes combined with in vivo subcellular
targeting verifi cation indicates novel metabolic
and regulatory functions of peroxisomes. Plant
Physiol 150:125-143
9. Fukao Y, Hayashi M, Hara-Nishimura I et al
(2003) Novel glyoxysomal protein kinase,
GPK1, identifi ed by proteomic analysis of gly-
oxysomes in etiolated cotyledons of Arabidopsis
thaliana. Plant Cell Physiol 44:1002-1012
10. Eubel H, Meyer EH, Taylor NL et al (2008)
Novel proteins, putative membrane transport-
ers, and an integrated metabolic network are
revealed by quantitative proteomic analysis of
Arabidopsis cell culture peroxisomes. Plant
Physiol 148:1809-1829
